CRISPR enabled multiplexed genome engineering

ABSTRACT

Described herein are method for generating a vector for editing a cell. The method comprises ligating into a vector that encodes a portion of a gRNA a cassette comprising at least one editing cassette, a promoter, and a gene encoding another portion of the gRNA. Upon ligation, the portion of the gRNA from the editing cassette and the other portion of the gRNA are ligated and form a functional gRNA.

RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 16/550,092, filed Aug. 23, 2019, which is acontinuation application of U.S. patent application Ser. No. 16/275,439,filed Feb. 14, 2019, now U.S. Pat. No. 10,435,715, which is acontinuation application of U.S. patent application Ser. No. 16/056,310,filed Aug. 6, 2018, now U.S. Pat. No. 10,364,442, which is acontinuation application of U.S. patent application Ser. No. 15/948,789,filed Apr. 9, 2018, now U.S. Pat. No. 10,240,167, and is a continuationapplication of U.S. patent application Ser. No. 15/948,785, filed Apr.9, 2018, now U.S. Pat. No. 10,351,877, which is a continuation of U.S.patent application Ser. No. 15/630,909, filed Jun. 22, 2017, now U.S.Pat. No. 9,982,278, which is continuation application of U.S. patentapplication Ser. No. 15/116,616, filed Aug. 4, 2016, now U.S. Pat. No.10,266,849, which is a national stage entry of International ApplicationNo. PCT/US2015/015476, filed Feb. 11, 2015, which claims the benefitunder 35 U.S.C. § 119 of U.S. Provisional Application 61/938,608 filedFeb. 11, 2014, the entire teachings of each of which are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 17, 2020, isnamed 14325_001US12-TR1_SL.txt and is 13,480 bytes in size.

The sequence listing has been amended to include the sequences presentin FIG. 15B of the application as filed.

BACKGROUND OF THE INVENTION

Rational manipulation of large DNA constructs is a central challenge tocurrent synthetic biology and genome engineering efforts. In recentyears, a variety of technologies have been developed to address thischallenge and increase the specificity and speed with which mutationscan be generated. Additionally, adaptive mutations are a central driverof evolution, but their abundance and relative contribution to cellularphenotypes are poorly understood even in the most well-studiedorganisms. This can be attributed in large part to the technicalchallenges associated with observing and reconstructing these genotypesand correlating their presence with the phenotype of interest. Forexample, methods of genome editing that rely on random mutagenesis leadto complex genotypes consisting of many mutations, the relativecontribution of each of which is difficult to deconvolute. Moreover,epistatic interactions between alleles are difficult to assign due tolack of information regarding the individual mutations.

SUMMARY OF THE INVENTION

Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)exist in many bacterial genomes and have been found to play an importantrole in adaptive bacterial immunity. Transcription of these arrays givesrise to CRISPR RNAs that direct sequence-specific binding of CRISPR/cascomplexes to DNA targets in cells for gene repression or DNA cleavage.The specificity of these complexes allows novel in vivo applications forstrain engineering.

Described herein are methods of rational, multiplexed manipulation ofchromosomes within open reading frames (e.g., to generate proteinlibraries) or within multiple genes in any segment of a chromosome, inwhich various CRISPR systems are used. These methods provide moreefficient combinatorial genome engineering than those previouslyavailable.

Expanding the multiplexing capabilities of CRISPR presents a currenttechnological challenge and would enable use of these systems togenerate rational libraries in high-throughput format. Such advanceshave broad reaching implications for the fields of metabolic and proteinengineering that seek to refactor complex genetic networks for optimalproduction.

The methods comprise introducing components of the CRISPR system,including CRISPR-associated nuclease Cas9 and a sequence-specific guideRNA (gRNA) into cells, resulting in sequence-directed double strandedbreaks using the ability of the CRISPR system to induce such breaks.Components of the CRISPR system, including the CRISPR-associatednuclease Cas9 and a sequence-specific guide RNA (gRNA), can beintroduced into cells encoded on one or more vector, such as a plasmid.DNA recombineering cassettes or editing oligonucleotides can berationally designed to include a desired mutation within a target locusand a mutation in a common location outside of the target locus that maybe recognized by the CRISPR system. The described methods can be usedfor many applications, including altering a pathway of interest.

In one embodiment, the method is a method of genome engineering,comprising: (a) introducing into cells a vector that encodes: (i) anediting cassette that includes a region which is homologous to thetarget region of the nucleic acid in the cell and includes a mutation(referred to a desired mutation) of at least one nucleotide relative tothe target region, such as a mutation of at least one nucleotide in atleast one codon relative to the target region, and a protospaceradjacent motif (PAM) mutation; (ii) a promoter; and (iii) at least oneguide RNA (gRNA), the gRNA comprising: (a) a region (RNA) complementaryto a portion of the target region; and (b) a region (RNA) that recruitsa Cas9 nuclease, thereby producing cells comprising the vector; (b)maintaining cells comprising the vector under conditions under whichCas9 is expressed, wherein Cas9 nuclease is encoded on the vector,encoded on a second vector or encoded on the genome of the cells,resulting in production of cells that comprise the vector and do notcomprise the PAM mutation and cells that comprise the vector and the PAMmutation; (c) culturing the product of (b) under conditions appropriatefor cell viability, thereby producing viable cells; (d) obtaining viablecells produced in (c); and (e) sequencing the editing oligonucleotide ofthe vector of at least one viable cell obtained in (d) and identifyingthe mutation of at least one codon.

In another embodiment, the method is a method of genome engineering bytrackable CRISPR enriched recombineering, comprising: (a) introducinginto a first population of cells a vector that encodes: (i) at least oneediting cassette comprising: (a) a region homologous to a target regionof a nucleic acid and comprising a mutation of at least one nucleotiderelative to the target region, such as a mutation of at least onenucleotide in at least one codon relative to the target region, and (b)a protospacer adjacent motif (PAM) mutation; (ii) at least one promoter;and (iii) at least one guide RNA (gRNA) comprising: (a) a region (RNA)complementary to a portion of the target region and (b) a region (RNA)that recruits a Cas9 nuclease, thereby producing a second population ofcells that comprise the vector; (b) maintaining the second population ofcells under conditions in which Cas9 nuclease is expressed, wherein theCas9 nuclease is encoded on the vector, a second vector or on the genomeof cells of the second population of cells, resulting in DNA cleavage incells that do not comprise the PAM mutation and death of such cells; (c)obtaining viable cells produced in (b); and (d) identifying the mutationof at least one codon by sequencing the editing oligonucleotide of thevector of at least one cell of the second population of cells.

Either of the above embodiments can further comprise synthesizing and/orobtaining a population of editing oligonucleotides. Either embodimentcan further comprise amplifying the population of editingoligonucleotides. In any of the embodiments, the vector can furthercomprise a spacer, at least two priming sites or both a spacer and atleast two priming sites. In some embodiments, the editing cassettecomprises a target region comprising a mutation of at least one codonwithin 100 nucleotides of the PAM mutation.

Also described is a vector comprising:

-   -   (i) an editing cassette that includes a region which is        homologous to a target region of a nucleic acid in a cell and        includes a mutation (referred to a desired mutation) of at least        one nucleotide relative to the target region, and a protospacer        adjacent motif (PAM) mutation;    -   (ii) a promoter; and    -   (iii) at least one guide RNA (gRNA) comprising: (a) a region        (RNA) complementary to a portion of the target region; and (b) a        region (RNA) that recruits a Cas9 nuclease.

A further embodiment is a vector comprising:

-   -   (i) an editing cassette that includes a region which is        homologous to a target region of a nucleic acid in a cell and        includes a mutation (referred to a desired mutation) of at least        one nucleotide in at least one codon relative to the target        region, and a protospacer adjacent motif (PAM) mutation;    -   (ii) a promoter; and    -   (iii) at least one guide RNA (gRNA) comprising: (a) a region        (RNA) complementary to a portion of the target region; and (b) a        region (RNA) that recruits a Cas9 nuclease.

A further embodiment is a vector comprising:

-   -   (i) at least one editing cassette comprising: (a) a region        homologous to a target region of a nucleic acid and comprising a        mutation of at least one nucleotide relative to the target        region and (b) a protospacer adjacent motif (PAM) mutation;    -   (ii) at least one promoter; and    -   (iii) at least one guide RNA (gRNA) comprising: (a) a region        (RNA) complementary to a portion of the target region and (b) a        region (RNA) that recruits a Cas9 nuclease.

Another embodiment of the vector is a vector comprising:

-   -   (i) at least one editing cassette comprising: (a) a region        homologous to a target region of a nucleic acid and comprising a        mutation of at least one nucleotide in at least one codon        relative to the target region and (b) a protospacer adjacent        motif (PAM) mutation;    -   (ii) at least one promoter; and    -   (iii) at least one guide RNA (gRNA) comprising: (a) a region        (RNA) complementary to a portion of the target region and (b) a        region (RNA) that recruits a Cas9 nuclease.

In any of the embodiments, the vector can further comprise a spacer; atleast two priming sites; or a spacer and at least two priming sites. Inthose vectors in which the mutation is of at least one nucleotide in atleast one codon, the editing cassette the mutation can be, for example,within 100 nucleotides of the PAM mutation.

Also described is a library comprising a population of cells produced bythe methods described herein. A library of a population of cells cancomprise cells having any of the vectors described herein. For example,a population of cells can comprise a vector that comprises:

-   -   (i) an editing cassette that includes a region which is        homologous to a target region of a nucleic acid in a cell and        includes a mutation (referred to a desired mutation) of at least        one nucleotide relative to the target region, and a protospacer        adjacent motif (PAM) mutation;    -   (ii) a promoter; and    -   (iii) at least one guide RNA (gRNA) comprising: (a) a region        (RNA) complementary to a portion of the target region; and (b) a        region (RNA) that recruits a Cas9 nuclease.

In a further embodiment, a population of cells can comprise a vectorthat comprises:

-   -   (i) an editing cassette that includes a region which is        homologous to a target region of a nucleic acid in a cell and        includes a mutation (referred to a desired mutation) of at least        one nucleotide in at least one codon relative to the target        region, and a protospacer adjacent motif (PAM) mutation;    -   (ii) a promoter; and    -   (iii) at least one guide RNA (gRNA) comprising: (a) a region        (RNA) complementary to a portion of the target region; and (b) a        region (RNA) that recruits a Cas9 nuclease.

In a further embodiment, the method is a method of CRISPR-assistedrational protein engineering (combinatorial genome engineering),comprising:

-   -   (a) constructing a donor library, which comprises recombinant        DNA, such as recombinant chromosomes or recombinant DNA in        plasmids, by introducing into, such as by co-transformation, a        population of first cells (i) one or more editing        oligonucleotides, such as rationally designed oligonucleotides,        that couple deletion of a first single protospacer adjacent        motif (PAM) with mutation of at least one codon in a gene        adjacent to the PAM (the adjacent gene) and (b) a guide RNA        (gRNA) that targets a nucleotide sequence 5′ of the open reading        frame of a chromosome, thereby producing a donor library that        comprises a population of first cells comprising recombinant        chromosomes having targeted codon mutations;    -   (b) amplifying the donor library constructed in (a), such as by        PCR amplification, of recombinant chromosomes that uses a        synthetic feature from the editing oligonucleotides and        simultaneously incorporates a second PAM deletion (destination        PAM deletion) at the 3′ terminus of the gene, thereby coupling,        such as covalently coupling, targeted codon mutations directly        to the destination PAM deletion and producing a retrieved donor        library carrying the destination PAM deletion and targeted codon        mutations; and    -   (c) introducing (e.g., co-transforming) the donor library        carrying the destination PAM deletion and targeted codon        mutations and a destination gRNA plasmid into a population of        second cells, which are typically a population of naïve cells,        thereby producing a destination library comprising targeted        codon mutations.

The population of first cells and the population of second cells (e.g.,a population of naïve cells) are typically a population in which thecells are all of the same type and can be prokaryotes or eukaryotes,such as but not limited to bacteria, mammalian cells, plant cells,insect cells.

In some embodiments, the method further comprises maintaining thedestination library under conditions under which protein is produced.

In some embodiments, the first cell expresses a polypeptide with Cas9nuclease activity. In some embodiments, the polypeptide with Cas9nuclease activity is expressed under control of an inducible promoter.

In some embodiments, the editing oligonucleotides are complementary to a(one, one or more, at least one) target nucleic acid present in thefirst cell. In some embodiments, the editing oligonucleotides targetmore than one target site or locus in the first cell. In someembodiments, the nucleic acid sequence of the editing oligonucleotides[desired codon] comprises one or more substitutions, deletions,insertions or any combination of substitutions, deletions and insertionsrelative to the target nucleic acid. In some embodiments, the editingoligonucleotides are rationally designed; in further embodiments, theyare produced by random mutagenesis or by using degenerate primeroligonucleotides. In some embodiments, the editing oligonucleotides arederived from a collection of nucleic acids (library).

In some embodiments, the gRNA is encoded on a plasmid. In someembodiments, the editing oligonucleotide and the gRNA are introducedinto the first cell by transformation, such as by co-transformation ofthe editing oligonucleotide and the guide (g)RNA. In some embodiments,the editing oligonucleotide and the gRNA are introduced sequentiallyinto the first cell. In other embodiments, the editing oligonucleotideand the gRNA are introduced simultaneously into the first cell.

In some embodiments, retrieving the donor library further comprises (a)screening cells for incorporation of the editing oligonucleotide and (b)selecting cells that are confirmed to have incorporated the editingoligonucleotide. In some embodiments, retrieving the donor libraryfurther comprises processing of the retrieved donor library.

In some embodiments, the destination cell/naïve cell expresses apolypeptide with Cas9 nuclease activity. In some embodiments, thepolypeptide with Cas9 nuclease activity is expressed under control of aninducible promoter.

Also described is a method of CRISPR-assisted rational proteinengineering, comprising:

-   -   (a) introducing (e.g., co-transforming) (i) synthetic dsDNA        editing cassettes comprising editing oligonucleotides and (ii) a        vector that expresses a guide RNA (gRNA) that targets genomic        sequence just upstream of a gene of interest into a population        of first cells, under conditions under which multiplexed        recombineering and selective enrichment by gRNA of the editing        oligonucleotides occur, thereby producing a donor library;    -   (b) amplifying the donor library with an oligonucleotide that        deletes a protospacer adjacent motif (PAM) adjacent to the 3′        end of the gene of interest (destination PAM), thereby producing        an amplified donor library comprising dsDNA editing cassettes        from which the destination PAM has been deleted (with a 3′ PAM        deletion), rational codon mutations, and a P1 site;    -   (c) processing the amplified donor library with an enzyme, such        as a restriction enzyme (e.g., BsaI), to remove the P1 site; and    -   (d) co-transforming a population of naïve cells with the        amplified donor library processed in (c) and destination gRNA,        thereby producing a population of co-transformed cells        comprising dsDNA editing cassettes from which the destination        PAM has been deleted (with a 3′ PAM deletion), rational codon        mutations and destination gRNA.

In all embodiments described, a mutation can be of any type desired,such as one or more insertions, deletions, substitutions or anycombination of two or three of the foregoing (e.g., insertion anddeletion; insertion and substitution; deletion and substitution;substitution and insertion; insertion, deletion and substitution).Insertions, deletions and substitutions can be of any number ofnucleotides. They can be in codons (coding regions) and/or in noncodingregions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B present an overview of CRISPR assisted rational proteinengineering (CARPE). FIG. 1A shows a schematic of donor libraryconstruction. Synthetic dsDNA editing cassettes were co-transformed witha vector that expresses a guide RNA (gRNA) targeting the genomicsequence upstream of the gene of interest. The co-transformationgenerated a donor library via multiplexed recombineering of the editingoligonucleotides, which are selectively enriched by the gRNA. The donorlibrary was then amplified using an oligonucleotide that mutates(deletes) a PAM adjacent to the 3′ end of the gene (destination PAM).FIG. 1B shows a schematic of final protein library generation. The donorlibrary was processed with BsaI to remove the P1 site, and the libraryof dsDNA cassettes with the 3′PAM deletion and rational codon mutationswas co-transformed with the destination gRNA to generate the finalprotein library.

FIG. 2 presents the DNA sequence from clones from the galK donor libraryconstruction confirming incorporation of the P1 feature of the editingoligonucleotide at high efficiency as well as the mutation at thetargeted codon position (underlined). The sequence of P1 is provided bySEQ ID NO: 1.

FIG. 3A shows primer design. FIG. 3B shows the expected density relativeto the number primers.

FIG. 4A presents linker and construct results. FIG. 4B shows 10 editsrelated to emulsion PCR based tracking.

FIG. 5 is a schematic of rational protein editing for metabolicengineering.

FIG. 6 is a schematic of the generation of CRISPR enriched rationalprotein libraries.

FIG. 7 is a schematic of setup and demonstration of CARPE.

FIG. 8 shows strategies for iterative CRISPR co-selection.

FIG. 9 presents a strategy for multiplexed protein engineering usingCARPE.

FIG. 10 shows construction of a galK donor library using CARPE.

FIG. 11A shows a schematic of multiplex CRISPR-based editing usingCARPE.

FIG. 11B shows a schematic of multiplex CRISPR-based editing usinggenome engineering by trackable CRISPR enriched recombineering(GEn-TraCER).

FIG. 12 shows a representative GEn-TraCER vector (construct) thatincludes an editing cassette for editing codon 24 of galK, a promoter,and spacer.

FIG. 13 shows the results of a galK editing using GEn-TraCER. The toppanels show DNA sequencing results of the chromosome and vector(plasmid) from cells that had been transformed with the galK codon 24editing GEn-TraCER vector, indicating the editing cassette(oligonucleotide) on the vector may be sequenced as a “trans-barcode”allowing high efficiency tracking of the desired genomic edit(mutation). The bottom panels show DNA sequencing chromatographs ofcells that exhibit the unedited, wild-type phenotype (red). The methodallows identification of cells with multiple chromosomes that carry boththe wild-type, unedited allele and the edited/mutated allele.

FIGS. 14A-14C show schematics of GEn-TraCER. FIG. 14A shows an overviewof the design components. The GEn-TraCER cassettes contain guide RNA(gRNA) sequence(s) to target a specific site in the cell genome andcause dsDNA cleavage. A region of homology complementary to the targetregion mutates the PAM and other nearby desired sites. Cells thatundergo recombination are selectively enriched to high abundance.Sequencing of the GEn-TraCER editing cassette in the vector enablestracking of the genomic edits/mutations. FIG. 14B shows an exampleediting cassette design for the E. coli galK gene at codon 145. The PAMis deleted with the nearest available PAM mutation that can be made forsynonymous change at the nearest available PAM position. This enablesmutagenesis with a “silent scar” of 1-2 nucleotides at the PAM deletionsite. FIG. 14C shows GEn-TraCER cassettes may be synthesized usingarray-based synthesis methods, thus enabling parallel synthesis of atleast 10⁴-10⁶ cassettes for systematic targeting and simultaneousevaluation of fitness for thousands of mutations on a genome-wide scale.

FIG. 15A shows an overview of GEn-TraCER vectors. FIG. 15B shows aportion of a representative GEn-TraCER for generation of a Y145*mutation in the E. coli galK gene in which the PAM mutation and thecodon that is mutated are separated by 17 nucleotides. The nucleic acidsequence of the portion of the representative GEn-TraCER is provided bySEQ ID NO: 28 and the reverse complement is provided by SEQ ID NO: 33.

FIGS. 16A-16C present controls for GEn-TraCER design. FIG. 16A shows theeffect of the size of the editing cassette on efficiency of the method.FIG. 16B shows the effect of the distance between the PAMmutation/deletion and the desired mutation on efficiency of the method.FIG. 16C shows the effect of the presence or absence of the MutS systemon efficiency of the method.

DETAILED DESCRIPTION OF THE INVENTION

Bacterial and archaeal CRISPR systems have emerged as powerful new toolsfor precision genome editing. The type-II CRISPR system fromStreptococcus pyogenes (S. pyogenes) has been particularly wellcharacterized in vitro, and simple design rules have been establishedfor reprogramming its double-stranded DNA (dsDNA) binding activity(Jinek et al. Science (2012) 337(6096): 816-821). Use of CRISPR-mediatedgenome editing methods has rapidly accumulated in the literature in awide variety of organisms, including bacteria (Cong et al. Science(2013) 339 (6121): 819-823), Saccharomyces cerevisiae (DiCarlo et al.Nucleic Acids Res. (2013) 41:4336-4343), Caenorhabditis elegans(Waaijers et al. Genetics (2013) 195: 1187-1191) and various mammaliancell lines (Cong et al. Science (2013) 339 (6121): 819-823; Wang et al.Cell (2013) 153:910-918). Like other endonuclease based genome editingtechnologies, such as zinc-finger nucleases (ZFNs), homing nucleases andTALENS, the ability of CRISPR systems to mediate precise genome editingstems from the highly specific nature of target recognition. Forexample, the type-I CRISPR system from Escherichia coli and the S.pyogenes system require perfect complementarity between the CRISPR RNA(crRNA) and a 14-15 base pair recognition target, suggesting that theimmune functions of CRISPR systems are naturally employed (Jinek et al.Science (2012) 337(6096): 816-821; Brouns et al. Science (2008)321:960-964; Semenova et al. PNAS (2011) 108:10098-10103).

Described herein are methods for genome editing that employ anendonuclease, such as the Cas9 nuclease encoded by a cas9 gene, toperform directed genome evolution/produce changes (deletions,substitutions, additions) in DNA, such as genomic DNA. The cas9 gene canbe obtained from any source, such as from a bacterium, such as thebacterium S. pyogenes. The nucleic acid sequence of the cas9 and/oramino acid sequence of Cas9 may be mutated, relative to the sequence ofa naturally occurring cas9 and/or Cas9; mutations can be, for example,one or more insertions, deletions, substitutions or any combination oftwo or three of the foregoing. In such embodiments, the resultingmutated Cas9 may have enhanced or reduced nuclease activity relative tothe naturally occurring Cas9.

FIGS. 1A, 1B, and 11A present a CRISPR-mediate genome editing methodreferred to as CRISPR Assisted Rational Protein Engineering (CARPE).CARPE is a two stage construction process which relies on generation of“donor” and “destination” libraries that incorporate directed mutationsfrom single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) editingcassettes directly into the genome. In the first stage of donorconstruction (FIG. 1A), rationally designed editing oligos arecotransformed into cells with a guide RNA (gRNA) that hybridizesto/targets a target DNA sequence, such as a sequence 5′ of an openreading frame or other sequence of interest. A key innovation of CARPEis in the design of the editing oligonucleotides that couple deletion ormutation of a single protospacer adjacent motif (PAM) with the mutationof one or more desired codons in the adjacent gene, thereby enablinggeneration of the entire donor library in a single transformation. Thedonor library is then retrieved by amplification of the recombinantchromosomes, e.g. by a PCR reaction, using a synthetic feature from theediting oligonucleotide; a second PAM deletion or mutation issimultaneously incorporated at the 3′ terminus of the gene. Thisapproach thus covalently couples the codon targeted mutations directlyto a PAM deletion. In the second stage of CARPE (FIG. 1B) the PCRamplified donor libraries carrying the destination PAM deletion/mutationand the targeted mutations (desired mutation(s) of one or morenucleotides, such as one or more nucleotides in one or more codons) areco-transformed into naïve cells with a destination gRNA vector togenerate a population of cells that express a rationally designedprotein library.

In the CRISPR system, the CRISPR trans-activating (tracrRNA) and thespacer RNA (crRNA) guide selection of a target region. As used herein, atarget region refers to any locus in the nucleic acid of a cell orpopulation of cells in which a mutation of at least one nucleotide, suchas a mutation of at least one nucleotide in at least one codon (one ormore codons), is desired. The target region can be, for example, agenomic locus (target genomic sequence) or extrachromosomal locus. ThetracrRNA and crRNA can be expressed as a single, chimeric RNA molecule,referred to as a single-guide RNA, guide RNA, or gRNA. The nucleic acidsequence of the gRNA comprises a first nucleic acid sequence, alsoreferred to as a first region, that is complementary to a region of thetarget region and a second nucleic acid sequence, also referred to asecond region, that forms a stem loop structure and functions to recruitCas9 to the target region. In some embodiments, the first region of thegRNA is complementary to a region upstream of the target genomicsequence. In some embodiments, the first region of the gRNA iscomplementary to at least a portion of the target region. The firstregion of the gRNA can be completely complementary (100% complementary)to the target genomic sequence or include one or more mismatches,provided that it is sufficiently complementary to the target genomicsequence to specifically hybridize/guide and recruit Cas9. In someembodiments, the first region of the gRNA is at least 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or at least 30 nucleotidesin length. In some embodiments, the first region of the gRNA is at least20 nucleotides in length. In some embodiments the stem loop structurethat is formed by the second nucleic acid sequence is at least 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 7, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides inlength. In specific embodiments, the stem loop structure is from 80 to90 or 82 to 85 nucleotides in length and, in further specificembodiments, the second region of the gRNA that forms a stem loopstructure is 83 nucleotides in length.

In some embodiments, the sequence of the gRNA (of the donor library)that is introduced into the first cell using the CARPE method is thesame as the sequence of the gRNA (of the destination library) that isintroduced into the second/naïve cell. In some embodiments, more thanone gRNA is introduced into the population of first cells and/or thepopulation of second cells. In some embodiments, the more than one gRNAmolecules comprise first nucleic acid sequences that are complementaryto more than one target region.

In the CARPE method, double stranded DNA cassettes, also referred to asediting oligonucleotides, for use in the described methods can beobtained or derived from many sources. For example, in some embodiments,the dsDNA cassettes are derived from a nucleic acid library that hasbeen diversified by nonhomologous random recombination (NRR); such alibrary is referred to as an NRR library. In some embodiments, theediting oligonucleotides are synthesized, for example by array-basedsynthesis. The length of the editing oligonucleotide may be dependent onthe method used in obtaining the editing oligonucleotide. In someembodiments, the editing oligonucleotide is approximately 50-200nucleotides, 75-150 nucleotides, or between 80-120 nucleotides inlength.

An editing oligonucleotide includes (a) a region that is homologous to atarget region of the nucleic acid of the cell and includes a mutation(referred to a desired mutation) of at least one codon relative to thetarget region, and (b) a protospacer adjacent motif (PAM) mutation. ThePAM mutation may be any insertion, deletion or substitution of one ormore nucleotides that mutates the sequence of the PAM such that it is nolonger recognized by the CRISPR system. A cell that comprises such a PAMmutation may be said to be “immune” to CRISPR-mediated killing. Thedesired mutation relative to the sequence of the target region may be aninsertion, deletion, and/or substitution of one or more nucleotides atat least one codon of the target region.

The CARPE method is described below with reference to a bacterial genefor purposes of illustration only. The methods may be applied to anygene(s) of interest, including genes from any prokaryote includingbacteria and archaea, or any eukaryote, including yeast and mammalian(including human) genes. The CARPE method was carried out on the galKgene in the E. coli genome, in part due to the availability of activityassays for this gene. The method was carried out using BW23115 parentalstrains and the pSIM5 vector (Datta et al. Gene (2008) 379:109-115) tomediate recombineering. The cas9 gene was cloned into the pBTBX-2backbone under the control of a pBAD promoter to allow control of thecleavage activity by addition of arabinose. Assessment of the ability toselectively incorporate synthetic dsDNA cassettes (127 bp) was carriedout using dsDNA cassettes from NNK libraries that were constructed fromdegenerate primers and/or from rationally designed oligonucleotides(oligos) synthesized as part of a 27,000 member library via microarraytechnology. In both cases, the oligonucleotides were designed to mutatethe active site residues of the galK gene product. Highly efficientrecovery of donor strain libraries was verified based on changes in theamplicon sizes obtained with primers directed at the galK locus.Sequencing of these colony PCR products from the NRR libraries indicatedthat the synthetic priming site (P1) from the dsDNA cassettes wasincorporated with about 90-100% efficiency. This indicated that theselibraries can be generated with high efficiency without reliance on theerror prone mutS knockout strains that have typically been used in otherrecombineering based editing approaches (Costantino et al. PNAS (2003)100:15748-15753; Wang et al. Nature (2009) 460:894-898). There was adrop in the efficiency of the codon mutations (about 20%), which may bedue to mutS corrections during allelic replacement. Preliminaryassessment of clones in the destination libraries indicated that thefinal codon editing efficiency was about 10% when both phases ofconstruction are carried out in the mutS⁺ background.

Comparison with other recently-published protocols for co-selectableediting was done, using alternative protocols that do not covalentlylink the PAM and codon mutations, but instead rely on their proximity toone another during replication (Wang et al. Nat. Methods (2012)9:591-593). In these non-covalent experiments the same editing oligos asabove were used and efforts were made to co-select for their insertionusing the ssDNA oligos that target the same donor/destination PAM sites.Colony screening of the resultant mutants reveals high efficiency inrecovery of the PAM mutants. However, there does not appear to be astrong co-selection for insertion of dsDNA editing cassettes. This maybe due to large differences in the relative recombineering efficienciesof the PAM deletion oligonucleotides and the editing cassettes whichgenerate sizable chromosomal deletions.

The ability to improve final editing efficiencies of the CARPE methodcan be assessed, such as by carrying out donor construction in mutSdeficient strains before transferring to a wild-type donor strain in aneffort to prevent loss of mutations during the donor construction phase.In addition, the generality of the CARPE method can be assessed, such asby utilizing CARPE on a number of essential genes, including dxs, metA,and folA. Essential genes have been effectively targeted using gRNAdesign strategies described. Results also indicate that despite the genedisruption that occurs during the donor library creation, the donorlibraries can be effectively constructed and retrieved within 1-3 hourspost recombineering.

Also provided herein are methods for trackable, precision genome editingusing a CRISPR-mediated system referred to as Genome Engineering byTrackable CRISPR Enriched Recombineering (GEn-TraCER). The GEn-TraCERmethods achieve high efficiency editing/mutating using a single vectorthat encodes both the editing cassette and gRNA. When used with parallelDNA synthesis, such as array-based DNA synthesis, GEN-TraCER providessingle step generation of thousands of precision edits/mutations andmakes it possible to map the mutation by sequencing the editing cassetteon the vector, rather than by sequencing of the genome of the cell(genomic DNA). The methods have broad utility in protein and genomeengineering applications, as well as for reconstruction of mutations,such as mutations identified in laboratory evolution experiments.

The GEn-TraCER methods and vectors combine an editing cassette, whichincludes a desired mutation and a PAM mutation, with a gene encoding agRNA on a single vector, which makes it possible to generate a libraryof mutations in a single reaction. As shown in FIG. 11B, the methodinvolves introducing a vector comprising an editing cassette thatincludes the desired mutation and the PAM mutation into a cell orpopulation of cells. In some embodiments, the cells into which thevector is introduced also encodes Cas9. In some embodiments, a geneencoding Cas9 is subsequently introduced into the cell or population ofcells. Expression of the CRISPR system, including Cas9 and the gRNA, inthe cell or cell population is activated; the gRNA recruits Cas9 to thetarget region, where dsDNA cleavage occurs. Without wishing to be boundby any particular theory, the homologous region of the editing cassettecomplementary to the target region mutates the PAM and the one or morecodon of the target region. Cells of the population of cells that didnot integrate the PAM mutation undergo unedited cell death due toCas9-mediated dsDNA cleavage. Cells of the population of cells thatintegrate the PAM mutation do not undergo cell death; they remain viableand are selectively enriched to high abundance. Viable cells areobtained and provide a library of targeted mutations.

The method of trackable genome editing using GEn-TraCER comprises: (a)introducing a vector that encodes at least one editing cassette, apromoter, and at least one gRNA into a cell or population of cells,thereby producing a cell or population of cells comprising the vector (asecond population of cells); (b) maintaining the second population ofcells under conditions in which Cas9 is expressed, wherein the Cas9nuclease is encoded on the vector, a second vector or on the genome ofcells of the second population of cells, resulting in DNA cleavage anddeath of cells of the second population of cells that do not comprisethe PAM mutation, whereas cells of the second population of cells thatcomprise the PAM mutation are viable; (c) obtaining viable cells; and(d) sequencing the editing cassette of the vector in at least one cellof the second population of cells to identify the mutation of at leastone codon.

In some embodiments, a separate vector encoding cas9 is also introducedinto the cell or population of cells. Introducing a vector into a cellor population of cells can be performed using any method or techniqueknown in the art. For example, vectors can be introduced by standardprotocols, such as transformation including chemical transformation andelectroporation, transduction and particle bombardment.

An editing cassette includes (a) a region, which recognizes (hybridizesto) a target region of a nucleic acid in a cell or population of cells,is homologous to the target region of the nucleic acid of the cell andincludes a mutation (referred to a desired mutation) of at least onenucleotide in at least one codon relative to the target region, and (b)a protospacer adjacent motif (PAM) mutation. The PAM mutation may be anyinsertion, deletion or substitution of one or more nucleotides thatmutates the sequence of the PAM such that the mutated PAM (PAM mutation)is not recognized by the CRISPR system. A cell that comprises such as aPAM mutation may be said to be “immune” to CRISPR-mediated killing. Thedesired mutation relative to the sequence of the target region may be aninsertion, deletion, and/or substitution of one or more nucleotides atat least one codon of the target region. In some embodiments, thedistance between the PAM mutation and the desired mutation is at least5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 nucleotides on the editing cassette In someembodiments, the PAM mutation is located at least 9 nucleotides from theend of the editing cassette. In some embodiments, the desired mutationis located at least 9 nucleotides from the end of the editing cassette.

In some embodiments, the desired mutation relative to the sequence ofthe target region is an insertion of a nucleic acid sequence. Thenucleic acid sequence inserted into the target region may be of anylength. In some embodiments, the nucleic acid sequence inserted is atleast 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, or at least 2000 nucleotides in length. In embodimentsin which a nucleic acid sequence is inserted into the target region, theediting cassette comprises a region that is at least 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 51, 52,53, 54, 55, 56, 57, 58, 59, or at least 60 nucleotides in length andhomologous to the target region.

The term “GEn-TraCER cassette” may be used to refer to an editingcassette, promoter, spacer sequence and at least a portion of a geneencoding a gRNA. In some embodiments, portion of the gene encoding thegRNA on the GEn-TraCER cassette encodes the portion of the gRNA that iscomplementary to the target region. In some embodiments, the portion ofthe gRNA that is complementary to the target region is at least 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or at least 30nucleotides in length. In some embodiments, the portion of the gRNA thatis complementary to the target region is 24 nucleotides in length. Insome embodiments, the GEn-TraCER cassette further comprising at leasttwo priming sites. In some embodiments, the priming sites may be used toamplify the GEn-TraCER cassette, for example by PCR. In someembodiments, the portion of the gRNA is that complementary to the targetregion is used as a priming site.

In the GEn-TraCER method, editing cassettes and GEn-TraCER cassettes foruse in the described methods can be obtained or derived from manysources. For example, in some embodiments, the editing cassette issynthesized, for example by array-based synthesis. In some embodiments,the GEn-TraCER cassette is synthesized, for example by array-basedsynthesis. The length of the editing cassette and/or GEn-TraCER cassettemay be dependent on the method used in obtaining the editing cassetteand/or the GEn-TraCER cassette. In some embodiments, the editingcassette is approximately 50-300 nucleotides, 75-200 nucleotides, orbetween 80-120 nucleotides in length. In some embodiments, theGEn-TraCER cassette is approximately 50-300 nucleotides, 75-200nucleotides, or between 80-120 nucleotides in length.

In some embodiments, the method also involves obtaining GEn-TraCERcassettes, for example by array-based synthesis, and constructing thevector. Methods of constructing a vector will be known to one ordinaryskill in the art and may involve ligating the GEn-TraCER cassette into avector. In some embodiments, the GEn-TraCER cassettes or a subset (pool)of the GEn-TraCER cassettes are amplified prior to construction of thevector, for example by PCR.

The cell or population of cells comprising the vector and also encodingCas9 are maintained or cultured under conditions in which Cas9 isexpressed. Cas9 expression can be controlled. The methods describedherein involve maintaining cells under conditions in which Cas9expression is activated, resulting in production of Cas9. Specificconditions under which Cas9 is expressed will depend on factors, such asthe nature of the promoter used to regulate Cas9 expression. In someembodiments, Cas9 expression is induced in the presence of an inducermolecule, such as arabinose. When the cell or population of cellscomprising Cas9-encoding DNA are in the presence of the inducermolecule, expression of Cas9 occurs. In some embodiments, Cas9expression is repressed in the presence of a repressor molecule. Whenthe cell or population of cells comprising Cas9-encoding DNA are in theabsence of a molecule that represses expression of Cas9, expression ofCas9 occurs.

Cells of the population of cells that remain viable are obtained orseparated from the cells that undergo unedited cell death as a result ofCas9-mediated killing; this can be done, for example, by spreading thepopulation of cells on culture surface, allowing growth of the viablecells, which are then available for assessment.

The desired mutation coupled to the PAM mutation is trackable using theGEn-TraCER method by sequencing the editing cassette on the vector inviable cells (cells that integrate the PAM mutation) of the population.This allows for facile identification of the mutation without the needto sequence the genome of the cell. The methods involve sequencing ofthe editing cassette to identify the mutation of one of more codon.Sequencing can be performed of the editing cassette as a component ofthe vector or after its separation from the vector and, optionally,amplification. Sequencing may be performed using any sequencing methodknown in the art, such as by Sanger sequencing.

The methods described herein can be carried out in any type of cell inwhich the CRISPR system can function (e.g., target and cleave DNA),including prokaryotic and eukaryotic cells. In some embodiments the cellis a bacterial cell, such as Escherichia spp. (e.g., E. coli). In otherembodiments, the cell is a fungal cell, such as a yeast cell, e.g.,Saccharomyces spp. In other embodiments, the cell is an algal cell, aplant cell, an insect cell, or a mammalian cell, including a human cell.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to or expressed in a cell. Thedesired sequence(s) can be included in a vector, such as by restrictionand ligation or by recombination. Vectors are typically composed of DNA,although RNA vectors are also available. Vectors include, but are notlimited to: plasmids, fosmids, phagemids, virus genomes and artificialchromosomes.

Vectors useful in the GEN-TraCER method comprise at least one editingcassette as described herein, a promoter, and at least one gene encodinga gRNA. In some embodiments more than one editing cassette (for example2, 3, 4, 5, 6, 7, 8, 9, 10 or more editing cassettes) are included onthe vector. In some embodiments, the more than one editing cassettes arehomologous with different target regions (e.g., there are differentediting cassettes, each of which is homologous with a different targetregion). Alternatively or in addition, the vector may include more thanone gene encoding more than one gRNA, (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10or more gRNAs). In some embodiments, the more than one gRNAs containregions that are complementary to a portion of different target regions(e.g., there are different gRNAs, each of which is complementary to aportion of a different target region).

In some embodiments, a GEn-TraCER cassette comprising at least oneediting cassette, a promoter and a gene encoding a portion of a gRNA areligated into a vector that encodes another portion of a gRNA. Uponligation, the portion of the gRNA from the GEn-TraCER cassette and theother portion of the gRNA are ligated and form a functional gRNA.

The promoter and the gene encoding the gRNA are operably linked. In someembodiments, the methods involve introduction of a second vectorencoding Cas9. In such embodiments, the vector may further comprise oneor more promoters operably linked to a gene encoding Cas9. As usedherein, “operably” linked means the promoter affects or regulatestranscription of the DNA encoding a gene, such as the gene encoding thegRNA or the gene encoding Cas9. The promoter can be a native promoter (apromoter present in the cell into which the vector is introduced). Insome embodiments, the promoter is an inducible or repressible promoter(the promoter is regulated allowing for inducible or repressibletranscription of a gene, such as the gene encoding the gRNA or the geneencoding Cas9), such as promoters that are regulated by the presence orabsence of a molecule (e.g., an inducer or a repressor). The nature ofthe promoter needed for expression of the gRNA may vary based on thespecies or cell type and will be recognized by one of ordinary skill inthe art.

In some embodiments, the method comprises introducing a separate vectorencoding Cas9 into the cell or population of cells before or at the sametime as introduction of the vector comprising at least one editingcassette as described herein, a promoter and at least one gRNA. In someembodiments, the gene encoding Cas9 is integrated into the genome of thecell or population of cells. The Cas9-encoding DNA can be integratedinto the cellular genome before introduction of the vector comprising atleast one editing cassette as described herein, a promoter, and at leastone gRNA or after introduction of the vector comprising at least oneediting cassette as described herein, a promoter, and at least one gRNA.Alternatively, a nucleic acid molecule, such as DNA-encoding Cas9, canbe expressed from DNA integrated into the genome. In some embodiments,the gene encoding Cas9 is integrated into the genome of the cell.

Vectors useful in the GEn-TraCER methods described herein may furthercomprise a spacer sequence, two or more priming sites or both a spacersequence and two or more priming sites. In some embodiments, thepresence of priming sites flanking the GEn-TraCER cassette allowsamplification of the editing cassette, promoter and gRNA nucleic acidsequences.

EXAMPLES Example 1: Using the CARPE Method to Edit galK

The CARPE approach was-carried out on the galactokinase gene, galK, inthe E. coli genome; there are many available assays to assess theactivity of the gene product. The experiments were carried out using E.coli BW23115 parental strain and the pSIM5 vector (Datta et al. Gene(2008) 379:109-115) to mediate recombineering. The gene encoding Cas9was cloned into the pBTBX-2 backbone under the control of a pBADpromoter to allow control of the Cas9 cleavage activity by addition ofarabinose to the culture medium.

First, the ability to selectively incorporate of synthetic dsDNAcassettes (127 bp) was tested. The synthetic dsDNA cassettes werederived from NNR libraries that were constructed from degenerate primersor from rationally designed oligos synthesized as part of a 27,000member library via microarray technology. In both cases, theoligonucleotides were designed to mutate the active site residues of thegalK gene product as well as contain the synthetic priming site, P1 (SEQID NO: 1). Highly efficient recovery of donor strain libraries wasverified based on changes in the amplicon sizes obtained by colony PCRusing primers directed at the galK locus. Sequencing of the colony PCRproducts from the NNR libraries indicated that the synthetic primingsite (P1) from the dsDNA cassettes was incorporated with about 90-100%efficiency (FIG. 2). This surprising and unexpected result suggests thatlibraries can be generated with high efficiency without reliance on theerror prone mutS-deficient strains that have typically been used inother recombineering-based editing approaches (Constantino, et al. PNAS(2003) 100:15748-15753; Wang et al. Nature (2009) 460: 894-898).However, there was a drop in the efficiency of the codon mutations(about 20%), which may be due to correction by MutS during allelicreplacement. In this work, the final codon editing efficiency was about10% when both phases of construction were carried out in the mutS+background.

To enhance the final editing efficiencies and generality of the CARPEmethod, the donor construction may be performed in mutS-deficientstrains before transferring to a mutS+ donor strain in an effort toprevent loss of mutations during the donor construction phase.

Example 2: Using the CARPE Method to Target Essential Genes

In order to test the generality of the CARPE approach, the method wasused, as described above, on a number of essential genes, including dxs,metA, and folA. Essential genes can be targeted using the gRNA designstrategies (FIG. 3).

Data from CARPE experiments targeting the dxs gene also suggest thatdespite the gene disruption that occurs during the donor librarycreation, it is possible to effectively construct and retrieve the donorlibraries within 1-3 hours post recombineering.

Example 3: Using the CARPE Method to Modulate Production of Isopentenol

The hunt for better biofuels for industrial manufacturing via bacterialproduction requires the ability to perform state of the art genomedesign, engineering, and screening for the desired product. Previously,we demonstrated the ability to individually modify the expression levelsof every gene in the E. coli genome (Warner et al. Nat. Biotechnol(2010) 28:856-862). This method, termed trackable multiplexrecombineering (TRMR), produced a library of about 8000genomically-modified cells (˜4000 over-expressed genes and ˜4000 knockeddown genes). This library was later screened under different conditions,which enabled deeper understanding of gene products' activities andresulted in better performing strains under these selections. TRMRallowed modification of protein expression for two levels (overexpressedand knocked down) but did not enable the modification of the openreading frame (ORF). Here, we aim to produce large libraries of ORFmodifications and engineering whole metabolic pathways for the optimalproduction of biofuels.

A major difficulty in producing such libraries, which are rationallydesigned (in contrast to random mutagenesis), is the insertionefficiency of the desired mutations into the target cells.Recombineering, the canonical method for genome modifications in E.coli, uses recombinant genes from Lambda phage to facilitate theinsertion of foreign DNA into the host genome. However, this processsuffers from low efficiencies and may be overcome either by adding anantibiotic resistance gene followed by selection (as in TRMR), or byrecursively inducing recombination events (i.e., by MAGE (Wang et al.Nature (2008) 460:894-898). The CARPE method described herein increasesthe recombineering efficiency involving the use of the CRISPR system toremove all non-recombinant cells from the population. CRISPR is arecently discovered RNA-based, adaptive defense mechanism of bacteriaand archaea against invading phages and plasmids (Bhaya et al. Ann. Rev.of Genetics (2011) 45:273-297). This system underwent massiveengineering to enable sequence-directed double strand breaks using twoplasmids; one plasmid coding for the CRISPR-associated nuclease Cas9 andthe second plasmid coding for the sequence-specific guide RNA (gRNA)that guides Cas9 to its unique location (Qi et al. Cell (2013)45:273-297). The CARPE method utilizes the CRISPR system's ability toinduce DNA breaks, and consequently cell death, in a sequence-dependentmanner. We produced DNA recombineering cassettes that, in addition tothe desired mutation within the ORF, include a mutation in a commonlocation outside of the open reading frame of the gene which is targetedby the CRISPR machinery. This approach of linking/coupling desiredmutations with the avoidance from CRISPR-mediated death, due to the PAMmutation/deletion, enables dramatic enrichment of the engineered cellswithin the total population of cells.

The method is further demonstrated using the DXS pathway. The DSXpathway results in the production of isopentenyl pyrophosphate (IPP)which results in the biosynthesis of terpenes and terpenoids.Interestingly, IPP can also be precursor of lycopene or isopentenol,given the addition of the required genes. While lycopene renders thebacterial colonies red, and hence is easily screenable, isopentenol isconsidered to be a ‘second generation’ biofuel with higher energydensity and lower water miscibility than ethanol. Three proteins wereselected for engineering: 1) DSX, the first and the rate-limiting enzymeof the pathway, 2) IspB, which diverts the metabolic flux from the DXSpathway, and 3) NudF, which has been shown to convert IPP to isopentenolin both E. coli and B. subtilis (Withers et al. App. Environ. Microbiol(2007) 73: 6277-6283; Zheng et al. Biotechnol. for biofuels (2013)6:57).Mutations in the genes encoding DXS and IspB will be screened forincreased lycopene production with a new image analysis tool developedfor colony color quantification. NudF activity will be assayed directlyby measuring isopentenol levels by GC/MS and indirectly by isopentenolauxotrophic cells that will serve as biosensors. This method providesthe ability to rationally engineer large mutational libraries into theE. coli genome with high accuracy and efficiency and a strain thatproduces high yield of isopentenol.

Example 4: Using the GEn-TraCER Method to Edit galK

The GEn-TraCER method was used to edit the galK gene, which has servedas a model system for recombineering in E. coli (Yu et al. 2000). Thefirst GEn-TraCER cassettes constructed were designed to introduce a stopcodon in place of an inframe PAM at codon 24 of galK, referred to asgalK_Q24 (FIG. 12). Constructs and vectors were designed using a custompython script to generate the requisite mutations in high throughput.

Control cassettes were cloned into the gRNA vector described by Qi etal. Cell (2013) using a the Circular Polymerase cloning (CPEC) method.The backbone was linearized with the following primers:CCAGAAATCATCCTTAGCGAAAGCTAAGGAT (SEQ ID NO: 29) andGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT (SEQ ID NO: 30).

GenTRACER cassettes were ordered as gblocks and amplified using thefollowing primers:

-   ATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTG G (SEQ    ID NO: 31),-   ACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC (SEQ ID    NO: 32).

The components were stitched together using CPEC and transformed into E.coli to generate the vectors. This procedure is to be performed inmultiplex using the pooled oligonucleotide libraries with cloningefficiencies on the order of 10⁴-10⁵ CFU/μg.

E. coli MG1655 cells carrying pSIM5 (lambda-RED plasmid) and the X2-cas9plasmid were grown to mid log phase (0.4-0.7 OD) at 30° C. in LB with 50μg/mL kanamycin and 34 μg/mL chloramphenicol. The recombineeringfunctions of the pSIM5 vector were induced at 42° C. for 15 min and thenplaced on ice for 10 min. Cells were then made electrocompetent bypelleting and washing 2× with 10 mL chilled H2O. Cells were transformedwith 100 ng of a GEn-TraCER plasmid (also encoding carbenicillinresistance) and recovered for 3 hrs at 37° C. 50-100 μL of cells wereplated to the appropriate media containing 50 μg/mL kanamycin and 100μg/ml carbenecillin to selectively enrich for the CRISPR-edited strains.Editing efficiencies for the galK gene were calculated using red/whitescreening on MacConkey agar supplemented with galactose.

Based on a screening on MacConkey agar editing efficiencies of ˜100%were observed with the galK_Q24* design. Interestingly, unlikeoligo-mediated recombineering methods that require mismatch repairknockouts to achieve high efficiency (Li et al. 2003; Sawitzke et al.2011; Wang et al. 2011), there was no effect in strains with or withoutthe mismatch repair machinery intact.

Chromosome and vector sequences were then verified by Sanger sequencing.

As anticipated the designed mutation in the vector was mirrored on thechromosome (FIG. 13) indicating that the mutation was present in bothlocations and that the plasmid serves as a transacting barcode(trans-barcode) or record of the genome edit.

The design was adapted for rational mutagenesis of protein coding frameson a genome scale by generating “silent selectable scars” that consistof synonymous PAM mutation (FIG. 14B, ΔPAM) to “immunize” the cellagainst Cas9-mediated cleavage but leave the translation productunperturbed. We reasoned that silent scars may allow co-selection fornearby edits at a codon or other feature of interest with highefficiency. The effects of the homology arm length and the distancebetween the PAM mutation/deletion and the desired mutation in galK wereassessed and the efficiencies compared (FIG. 16B). A significantincrease in mutational efficiency at the galK position 145 was observedwhen the homology arm length was extended from 80 to 100 nucleotides(˜5% and 45%, respectively) with identical PAM edits.

Example 5: Using the GEn-TraCER Method to Reconstruct Mutations

The GEn-TraCER approach was extended to a genomic scale using a customautomated design software that allows targeting of sites around thegenome with a simple user input definition. The approach was tested byreconstructing all of non-synonymous point mutations from a recentlyreported study of thermal adaptation in E. coli (Tenaillon et al. 2012).This study characterized the complete set of mutations that occurred in115 isolates from independently propagated strains. This datasetprovides a diverse source of mutations whose individual fitness effectsshed further light on the mechanistic underpinnings of this complexphenotype. Each of these mutations were reconstructed with a 2-foldredundancy in the codon usage and ΔPAM, where possible, to enablestatistical correction for both the PAM and target codon mutations indownstream fitness analysis.

Example 6: Using the GEn-TraCER Method to Modulate Genetic Interactions

A promoter rewiring library is generated by integrating a promoter thatis dynamically regulated by an environmental cue (oxygen level, carbonsource, stress) upstream of each gene in the E. coli genome. Using theGEn-TraCER method, strains are generated with rewired genotypes that maybe beneficial, for example for tolerance to chemicals of interest forproduction.

What is claimed is:
 1. A method for generating a vector for editing acell comprising: obtaining a vector that comprises a nucleotide sequenceencoding a first region of a gRNA; and ligating into the vector thatencodes the first region of the gRNA a second nucleotide sequenceencoding: a region homologous to a target region in a cell comprising achange in sequence relative to the target region; a site conferringimmunity to nuclease-mediated editing; a promoter; and a gene encoding asecond region of the gRNA, whereupon the ligation forms a functionalgRNA; thus generating the vector for editing the cell.
 2. The method ofclaim 1, wherein the first region of the gRNA comprises a nucleotidesequence encoding a tracrRNA.
 3. The method of claim 2, wherein thesecond region of the gRNA comprises a nucleotide sequence encoding acrRNA.
 4. The method of claim 1, wherein the first region is at least15-30 nucleotides in length.
 5. The method of claim 1, wherein the firstregion is at least 20 nucleotides in length.
 6. The method of claim 1,wherein said functional gRNA is a chimeric guide RNA.
 7. The method ofclaim 1, wherein the promoter and the nucleotide sequence encoding thefunctional gRNA are operably linked.
 8. The method of claim 1, whereinthe vector further comprises a nucleotide sequence encoding anendonuclease.
 9. The method of claim 1, wherein said first region of thegRNA comprises a nucleotide sequence that interacts with, or recruits,an endonuclease.
 10. The method of claim 1, wherein the site conferringimmunity to nuclease-mediated editing comprises a protospacer adjacentmotif (PAM) mutation.
 11. The method of claim 1, wherein the cell is abacterial cell.
 12. The method of claim 1, wherein the cell is a yeastcell.
 13. The method of claim 1, wherein the cell is a mammalian cell.14. The method of claim 1, wherein the target region is within anon-coding region.
 15. The method of claim 1, wherein the target regionis within a coding region.
 16. The method of claim 1, wherein the changein sequence relative to the target region comprises one or moresubstitutions relative to the target sequence.
 17. The method of claim1, wherein the change in sequence relative to the target regioncomprises one or more insertions relative to the target sequence. 18.The method of claim 1, wherein the change in sequence relative to thetarget region comprises one or more deletions relative to the targetsequence.
 19. A vector library comprising a plurality of vectors createdwith the method of claim
 1. 20. The vector library of claim 19, whereinthe plurality ofvectors encode a plurality of functional gRNAs thattarget at least two different target regions.